阅读说明：本技术 一种包含植酸酶突变体的重组载体或重组菌株的应用 (Application of recombinant vector or recombinant strain containing phytase mutant ) 是由 王兴吉 郭庆文 张�杰 盛花开 刘文龙 于 2017-09-30 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种包含植酸酶突变体的重组载体或重组菌株。本发明通过PCR获得来自枯草芽孢杆菌突变菌的耐热植酸酶突变基因A-2,并将A-2构建到pPIC9质粒上,得到重组质粒pPIC9-A-2,并转化到毕赤酵母GS115,筛选得到高表达耐热植酸酶的重组菌,所表达的耐热植酸酶在80℃保温处理30min,相对活性仍剩余75％左右,90℃处理30min,酶活仍存留25％以上,耐热性良好,可以广泛用于食品、饲料等行业。(The invention belongs to the technical field of biology, and particularly relates to a recombinant vector or a recombinant strain containing a phytase mutant. The invention obtains the heat-resistant phytase mutant gene A-2 from bacillus subtilis mutant bacteria through PCR, constructs the A-2 on pPIC9 plasmid to obtain recombinant plasmid pPIC9-A-2, converts the recombinant plasmid into pichia GS115, screens to obtain the recombinant bacteria of high-expression heat-resistant phytase, and the expressed heat-resistant phytase is heat-insulated at 80 ℃ for 30min, the relative activity is still about 75 percent, and is processed at 90 ℃ for 30min, the enzyme activity is still more than 25 percent, the heat resistance is good, and the invention can be widely used in industries of food, feed and the like.)

1. The application of the recombinant vector or the recombinant strain is characterized in that the recombinant vector or the recombinant strain comprises a phytase mutant coding gene shown in a sequence table SEQ ID No. 3.

2. The use according to claim 1, wherein the recombinant vector is obtained by inserting the gene represented by SEQ ID No.3 into an expression vector pPIC 9.

3. The use of claim 1, wherein the recombinant strain is obtained by expressing the gene shown in SEQ ID No.3 in the host cell Pichia pastoris GS 115.

4. Use according to claim 1 for the production of phytase.

5. The use according to claim 4, characterized in that the phytase is produced by a method comprising:

taking pPIC9 as an expression vector, taking pichia pastoris GS115 as a host cell, and taking a strain expressing the phytase mutant coding gene shown in SEQ ID No.3 as a production strain;

inoculating the strain in BMGY culture medium, culturing at 30 deg.C and 220r/min until OD600 is 10, centrifuging, collecting thallus, re-suspending the thallus in BMMY culture medium, and continuously culturing at 30 deg.C and 220r/min for 48 hr to obtain phytase;

the BMGY culture medium comprises 1% of yeast extract, 2% of peptone, 0.1mol/L phosphate buffer solution with pH of 6.0, 1.34% of YNB, 4 × 10^-5% biotin, 1% glycerol;

the BMMY culture medium comprises 1% of yeast extract, 2% of peptone, 0.1mol/L phosphate buffer solution with pH of 6.0, 1.34% of YNB, 4 × 10^-5% biotin, 0.5% methanol.

The technical field is as follows:

the invention relates to the technical field of biology, in particular to a recombinant vector or a recombinant strain containing phytase mutants.

Background art:

phosphorus is an essential mineral element of an animal body, most of phosphorus in plant tissues exists in the form of phytic acid (phytic acid) or phytate, phytic acid molecules can chelate metal ions and exist in the form of chelate, the solubility of the phytic acid molecules is low, the phytic acid molecules are equivalent to anti-nutritional factors, the phytic acid molecules are difficult to be absorbed by monogastric animals, and the phosphorus which is not fully utilized enters a water body through animal excrement to finally cause water body eutrophication.

The phytase is a general term of enzymes which catalyze the hydrolysis of phytic acid and salts thereof into inositol and phosphate, and the phytase is added into animal feed, so that the utilization rate of phosphorus in the feed can be improved, and the pollution of the discharge of the phosphorus to the environment can be reduced. In 1996, the FDA confirmed that phytase was safe for use in food and could be used in animal feed, and has become the third largest feeding enzyme. The phytase can be synthesized in a large amount by microorganisms and added into feed for animals to use so as to solve the problem that monogastric animals lack the phytase capable of utilizing phytate, but the heat treatment in the feed processing process requires that the phytase has certain heat resistance. The major problems faced at present are high production cost of phytase, low phytase yield and insufficient thermostability.

The invention content is as follows:

the invention aims to provide a phytase mutant with improved heat resistance and a pichia pastoris engineering strain thereof. The phytase mutant is from a bacillus subtilis BS2 obtained by ultraviolet mutagenesis, and the phytase mutant coding gene A-2 is cloned from BS2 and constructed into a recombinant plasmid, and then is expressed in pichia pastoris GS115, so that a pichia pastoris engineering strain is obtained.

The following definitions are used in the present invention:

1. nomenclature for amino acid and DNA nucleic acid sequences

The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification of Phytase mutants

"amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the mutant. E.g., Gln166Asn, indicating the substitution of the amino acid at position 166 from wild-type Gln to Asn, the numbering of the positions corresponding to the numbering of the amino acid sequence of the wild-type phytase of SEQ ID No. 2; "bases substituted by the original base position" is likewise used to indicate the mutated base in the mutant, the numbering of the position corresponding to the numbering of the nucleotide sequence of the wild-type phytase in SEQ ID No. 1.

In the present invention, A-1 represents a gene encoding a wild-type phytase, and A-2 represents a gene encoding a phytase mutant, the information being shown in the following table.

The amino acid sequence of the phytase mutant is shown in a sequence table SEQ ID No. 4;

the nucleotide sequence of the phytase mutation coding gene A-2 is shown in SEQ ID No. 3;

the expression vector for expressing the phytase is pPIC9, and the microbial host cell for transforming the expression vector is Pichia pastoris GS 115;

the pichia pastoris engineering bacteria are obtained by connecting phytase mutation coding gene A-2 with an expression vector pPIC9 and expressing in pichia pastoris GS 115;

the invention also provides application of the phytase mutant in the field of feed.

The experimental steps of the invention are as follows:

1. carrying out enzyme digestion on the coding gene A-2 of the phytase mutant, and connecting the phytase mutant to an expression vector pPIC9 to obtain a recombinant vector;

2. transforming the recombinant vector into pichia pastoris GS115 to obtain a production strain GS115/pPIC 9-A-2 of the phytase mutant;

3. GS115/pPIC 9-A-2 is used as a production strain for producing phytase by fermentation.

The enzymatic properties of the phytase mutant are as follows:

(1) pH 4-6, the enzyme activity is stable, and the optimum action pH is 5.0.

(2) Temperature: the enzyme activity is stable at 50-80 ℃, and the optimal action temperature is 70 ℃.

(3) Heat resistance: the enzyme still has over 75 percent of enzyme activity after being insulated for 30min at 80 ℃, and the enzyme activity still remains over 25 percent after being insulated for 30min at 90 ℃.

Has the advantages that:

1. the invention discloses a brand-new phytase mutant which has the characteristics of high enzyme activity and good thermal stability. The enzyme activity of the mutant fermentation liquor can reach 359U/mL, and is improved by 200 percent compared with the wild phytase;

2. the phytase obtained by the invention still has over 75 percent of enzyme activity after being preserved for 30min at 80 ℃, and the enzyme activity still remains over 25 percent after being preserved for 30min at 90 ℃.

Description of the drawings:

FIG. 1 shows a PCR identification electrophoretogram of a colony;

wherein M is marker, and lane 1 is gene A-2;

FIG. 2 Phytase optimum pH curve;

FIG. 3 phytase optimum temperature curves;

FIG. 4 temperature stability curve of phytase.

The specific implementation mode is as follows:

the present invention is described in more detail below with reference to specific examples, which are provided by way of illustration only and are not intended to limit the scope of the invention. Modifications may be made by those skilled in the art, which would be within the principles of the invention, and such modifications are to be considered within the scope of the invention. The molecular biological experiment method not specifically described in this example can be referred to "molecular cloning Experimental Manual".